Alternative Splicing Switches Potassium Channel Sensitivity to Protein Phosphorylation

Abstract

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.Large conductance calcium-and voltage-activated potassium (BK) 1 channels link intracellular chemical signaling events with the electrical properties of excitable cells in the endocrine, nervous, and vascular systems (1-3). BK channels are further potently modulated by reversible protein phosphorylation (4 -7). In native tissues BK channels display considerable diversity and plasticity in their regulation by reversible protein phosphorylation. For example, cAMP-dependent protein kinase (PKA) phosphorylation activates BK channels in smooth muscle cells and many neurones but inhibits channel activity in endocrine cells of the anterior pituitary (5, 7-11). Furthermore, the direction of channel regulation by PKA can be modified during challenges to homeostasis (9 -11).The pore-forming ␣-subunits of BK channels are derived from a single gene (Slo) that undergoes extensive alternative splicing to produce channels with distinct phenotypes (12-15). Importantly, alternative splicing of the ␣-subunit is dynamically regulated in adults, for example during stress or pregnancy (15, 16). Thus the diversity and plasticity of responses to PKA-dependent protein phosphorylation observed between BK channels in native tissues may result either from differential modulation of alternatively spliced BK channel ␣-subunits (12-15) or through their interaction with different signaling complexes and ␤-subunits (17-19).To address whether BK channel alternative splice variants are differentially regulated by PKA-mediated protein phosphorylation, we have examined the regulation of three mouse (mslo) BK channel variants (20 -22) expressed in HEK293 cells. BK channels are regulated by multiple protein kinase signaling pathways (5,19,23,24). We have thus assayed the functional regulation of BK channel splice variants by directly activating PKA that remains closely associated with the channels in excised inside-out patches. EXPERIMENTAL PROCEDURESMolecular and Cell Biology-cDNAs encod...

Control of alternative pre-mRNA splicing by Ca++ signals

“…Inclusion of these alternative exons regulates the subcellular localization of proteins or their functions [25,26,119,120,[127][128][129]123,125,134,[137][138][139][140][141], ranging from subtle changes such as current kinetics of ion channels to on or off switches in the sensitivity to protein kinases or hormones [26,114,139,[142][143][144] (Table 1). These observations suggest that the regulation of alternative splicing by Ca ++ signals has effects on the expression of genes involved in a variety of cellular functions, from cell adhesion molecules at the synapse, ion channels in the cellular endoplasmic membrane, caspase in the cytosol as well as splicing factors inside the nucleus.…”

“…The STREX-encoded peptide lies in the Ca ++ -sensitivity domain at the COOH-terminal [30,152]. Inclusion of this exon confers higher voltage and Ca ++ sensitivity on the channel [24,[29][30][31]153], inhibition by PKA and oxidation [142,154], and sensitivity to glucocorticoids [143]. The exon is included in endocrine cells and neurons and enriched in the high frequency region of cochleae [22][23][24]84,107], contributing to the fine-tuning of hearing frequencies in birds and turtles [23,31].…”

Leucine Zipper Domain Targets cAMP-dependent Protein Kinase to Mammalian BK Channels

“…Variant and Mutant cDNA Constructs-The cloning and sub-cloning of the mouse BK channel splice variants ZERO and STREX into the mammalian expression vector pcDNA3 or pcDNA3.1ϩ (Invitrogen) have been described previously (17,25). A C-terminal hemagglutinin (HA) tag was introduced into each channel construct by replacing the normal stop codon with a sequence encoding the HA tag from a mouse BK channel construct kindly provided by Dr. Yi Zhou and Prof. Irwin B Levitan.…”

“…In Drosophila, the catalytic subunit, but not the holoenzyme, of cAMP-dependent protein kinase (PKA) 1 binds directly to the intracellular C terminus (3,19) of the channel. Although PKA co-immunoprecipitates with mammalian BK channels in a splice variant-independent manner, the mechanism of complex assembly is unknown (17).…”

“…Phenotypic variation in native BK channels results from extensive alternative exon splicing of the ␣-subunit (6, 9, 10) as well as through interaction with regulatory ␤-subunits and accessory proteins (11)(12)(13). The BK channel ␣-subunit is a target for regulation by multiple protein kinases and protein phosphatases (3, 14 -18), and several protein kinases have been reported to co-immunoprecipitate with mammalian BK channels (3,16,17). Although several consensus phosphorylation sites have been identified by mutagenesis within the intracellular C-terminal domain, BK channel ␣-subunits do not contain previously identified protein-protein interaction domains.…”